LOCUS       sequence-215036-        6687 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression in E.Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6687)
  AUTHORS   Chang A, Abderemane-Ali F, Hura GL, Rossen ND, Gate RE, Minor DL Jr.
  TITLE     A Calmodulin C-Lobe Ca(2+)-Dependent Switch Governs Kv7 Channel 
            Function.
  JOURNAL   Neuron. 2018 Feb 21;97(4):836-852.e6. doi: 
            10.1016/j.neuron.2018.01.035. Epub 2018 Feb 8.
  PUBMED    29429937
REFERENCE   2  (bases 1 to 6687)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.neuron.2018.01.035"; journalName: "Neuron"; date: 
            "2018-02-21- 21"; volume: "97"; issue: "4"; pages: "836-852.e6"
COMMENT     Sequence Label: Kv7.4 AB (325-557, Delta 368-523)/pET28HMT
FEATURES             Location/Qualifiers
     source          1..6687
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     33..52
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        33..51
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    52..76
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             91..113
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             132..149
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             159..1259
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1233..1256
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1314..1334
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1584..1601
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1654..1672)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1668..1715
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1752..2207
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1839..1858)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2049..2070
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2299..3114)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2299..3114)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3022..3041
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3106..3125
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3236..3824
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3725..3744
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3978..3995
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4010..4149
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4075..4094)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4235..4257
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4251..4442)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5251..6333)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6295..6314
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6334..6411)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6617..6636
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
       61 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca
      121 tgggttcttc tcaccatcac catcaccatg gttcttctat gaaaatcgaa gaaggtaaac
      181 tggtaatctg gattaacggc gataaaggct ataacggtct cgctgaagtc ggtaagaaat
      241 tcgagaaaga taccggaatt aaagtcaccg ttgagcatcc ggataaactg gaagagaaat
      301 tcccacaggt tgcggcaact ggcgatggcc ctgacattat cttctgggca cacgaccgct
      361 ttggtggcta cgctcaatct ggcctgttgg ctgaaatcac cccggacaaa gcgttccagg
      421 acaagctgta tccgtttacc tgggatgccg tacgttacaa cggcaagctg attgcttacc
      481 cgatcgctgt tgaagcgtta tcgctgattt ataacaaaga tctgctgccg aacccgccaa
      541 aaacctggga agagatcccg gcgctggata aagaactgaa agcgaaaggt aagagcgcgc
      601 tgatgttcaa cctgcaagaa ccgtacttca cctggccgct gattgctgct gacgggggtt
      661 atgcgttcaa gtatgaaaac ggcaagtacg acattaaaga cgtgggcgtg gataacgctg
      721 gcgcgaaagc gggtctgacc ttcctggttg acctgattaa aaacaaacac atgaatgcag
      781 acaccgatta ctccatcgca gaagctgcct ttaataaagg cgaaacagcg atgaccatca
      841 acggcccgtg ggcatggtcc aacatcgaca ccagcaaagt gaattatggt gtaacggtac
      901 tgccgacctt caagggtcaa ccatccaaac cgttcgttgg cgtgctgagc gcaggtatta
      961 acgccgccag tccgaacaaa gagctggcaa aagagttcct cgaaaactat ctgctgactg
     1021 atgaaggtct ggaagcggtt aataaagaca aaccgctggg tgccgtagcg ctgaagtctt
     1081 acgaggaaga gttggcgaaa gatccacgta ttgccgccac tatggaaaac gcccagaaag
     1141 gtgaaatcat gccgaacatc ccgcagatgt ccgctttctg gtatgccgtg cgtactgcgg
     1201 tgatcaacgc cgccagcggt cgtcagactg tcgatgaagc cctgaaagac gcgcagacta
     1261 attcgagctc gaacaacaac aacaataaca ataacaacaa cctcgggatc gaggaaaacc
     1321 tgtattttca gggccatatg aaggtccagg agcagcaccg gcagaagcac ttcgagaagc
     1381 ggaggatgcc ggcagccaac ctcatccagg ctgcctggcg cctgtactcc accgatatga
     1441 gccgggccta cctgacagcc acctggtaca agcttgacga catcatgcct gctgtgaaga
     1501 cagtcatccg ctccatcagg attctcaagt tcctggtggc caaaaggaaa ttcaaggaga
     1561 cactgcgacc gtacgactaa tcacaccacc accaccacca ctgagatccg gctgctaaca
     1621 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     1681 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     1741 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     1801 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     1861 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     1921 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     1981 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     2041 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     2101 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     2161 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     2221 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2281 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     2341 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     2401 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     2461 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     2521 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     2581 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     2641 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
     2701 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     2761 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     2821 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     2881 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     2941 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     3001 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     3061 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     3121 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     3181 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3241 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3301 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3361 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     3421 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3481 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3541 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3601 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3661 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3721 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3781 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3841 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     3901 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     3961 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     4021 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     4081 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     4141 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     4201 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     4261 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     4321 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     4381 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     4441 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     4501 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     4561 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     4621 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     4681 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     4741 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     4801 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     4861 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     4921 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     4981 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     5041 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     5101 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     5161 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     5221 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5281 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5341 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     5401 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     5461 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     5521 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     5581 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     5641 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     5701 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     5761 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     5821 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     5881 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     5941 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     6001 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     6061 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     6121 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     6181 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     6241 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     6301 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     6361 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     6421 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     6481 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     6541 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     6601 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     6661 cggtgatgcc ggccacgatg cgtccgg
//
